Pluripotent stem cells have generated tremendous interest in the biomedical community. With the realization that stem cells can be isolated from many adult tissues has come the hope that cultures of relatively pure stem cells can be maintained in vitro for use in treating a wide range of conditions. Stem cells, with their capability for self-regeneration in vitro and their ability to produce differentiated cell types, may be useful for replacing the function of aging or failing cells in nearly any organ system. By some estimates, over 100 million Americans suffer from disorders that might be alleviated by tranplantation technologies that utilize stem cells (Perry (2000) Science 287:1423). Such illnesses include, for example, cardiovascular diseases, autoimmune diseases, diabetes, osteoporosis, cancers and burns.
Insulin-dependent diabetes (IDDM) is a good example of a disease that could be cured or ameliorated through the use of stem cells. Insulin-dependent diabetes mellitus is a disease characterized by elevated blood glucose and the absence of the hormone insulin. The cause of the raised sugar levels is insufficient secretion of the hormone insulin by the pancreas. In the absence of this hormone, the body's cells are not able to absorb sugar from the blood stream in normal fashion, and the excess sugar accumulates in the blood. Chronically elevated blood glucose damages tissues and organs. IDDM is treated with insulin injections. The size and timing of insulin injections are influenced by measurements of blood sugar.
There are over 400 million diabetics in the world today. For instance, diabetes is one of the most prevalent chronic diseases in the United States, and a leading cause of death. Estimates based on the 1993 National Health Interview Survey (NHIS) indicate that diabetes has been diagnosed in 1% of the U.S. population age <45 years, 6.2% of those age 45-64 years, and 10.4% of those age >65 years. In other terms, in 1993 an estimated 7.8 million persons in the United States were reported to have this chronic condition. In addition, based on the annual incidence rates for diabetes, it is estimated that about 625,000 new cases of diabetes are diagnosed each year, including 595,000 cases of non-insulin-dependent diabetes mellitus (NIDDM) and 30,000 cases of insulin-dependent diabetes mellitus (IDDM). Persons with diabetes are at risk for major complications, including diabetic ketoacidosis, end-stage renal disease, diabetic retinopathy and amputation. There are also a host of less directly related conditions, such as hypertension, heart disease, peripheral vascular disease and infections, for which persons with diabetes are at substantially increased risk.
While medications such as injectable insulin and oral hypoglycemics allow diabetics to live longer, diabetes remains the third major killer, after heart disease and cancer. Diabetes is also a very disabling disease, because medications do not control blood sugar levels well enough to prevent swinging between high and low blood sugar levels, with resulting damage to the kidneys, eyes, and blood vessels.
Replenishment of functional glucose-sensing, insulin-secreting pancreatic beta cells through islet transplantation has been a longstanding therapeutic target. The limiting factor in this approach is the availability of an islet source that is safe, reproducible, and abundant. Current methodologies use either cadaverous material or porcine islets as transplant substrates (Korbutt et al., 1997). However, significant problems to overcome are the low availability of donor tissue, the variability and low yield of islets obtained via dissociation, and the enzymatic and physical damage that may occur as a result of the isolation process (reviewed by Secchi et al., 1997; Sutherland et al., 1998). In addition are issues of immune rejection and current concerns with xenotransplantation using porcine islets (reviewed by Weir & Bonner-Weir, 1997).
As a further example, stem cells capable of generating blood cells would also be of tremendous value for treatment of several diseases. A number of diseases or conditions result frown inappropriate levels or inadequate function of blood platelets. For example, “thrombocytopenias” are the result of an abnormally small number of platelets in the circulating blood. Thombocytopenia can be due to antibody mediated platelet destruction, massive blood transfusions, cardio-pulmonary by-pass or bone marrow failure from malignant infiltration, aplastic anemia or chemotherapy. “Thrombocythemic” disorders, on the other hand, are the result of a high platelet count. Finally, “thrombocytopathic” blood disorders are characterized by abnormally low or high platelet function, although platelet counts may be normal. Blood platelets are required for the maintenance of normal hemostasis. Platelets initiate blood clot formation and release growth factors that speed the process of wound healing as well as potentially serving other functions. Blood platelets are circulating cells that are crucial for the prevention of bleeding and for blood coagulation. Megakaryocytes are the cellular source of platelets and arise from a common bone marrow precursor cell which gives rise to all hematopoietic cell lineages. Stem cells could be used to generate cells in vitro or could be implanted to provide a stable source of cells capable of producing platelets.
In addition, extensive radiation therapy is used to treat many cancers. The radiation is lethal to the patient's endogenous bone marrow stem cells. Currently, these are replaced by transplantation in a procedure fraught with complications. An abundant supply of hematopoietic stem cells could be used for repeated treatments to replenish the depleted endogenous cells.
Many neural disorders are marked by death of nerve cells. Adult nerve cells regenerate poorly and nerve death often causes irreparable damage to congnitive and sensorimotor functions. There has been some success in treating disorders caused by nerve death with transplants of fetal nerve tissue. Fetal tissue has a greater ability to take up residence in the adult brain and differentiate into the appropriate cell type. However, obtaining sufficient fetal tissue is difficult and presents many ethical problems. Neural stem cells are capable of differentiating into many cell types of the nervous system. Remarkably, some neural stem cells are capable of migrating through the brain and settling in regions of nerve cell death. Such cells may then generate new neural processes to integrate with the endogenous neural network. It is expected that neural stem cells can be used to treat disorders such as Alzheimer's disease, Parkinson's disease, stroke, ischemia, trauma, spinal cord injuries, damage from infectious disease etc.
It is an object of the present invention to provide simple methods for the isolation and propagation of stem cells from virtually any tissue type. Such stem cells can then be used, for example, for direct transplantation or to produce differentiated cells in vitro for transplantation or. The invention accordingly provides, for example, pancreatic and hepatic stem cells that may serve as a source for many other, more differentiated cell types such as pancreatic beta cells. Advantages lie in obviating the need for physical dissociation of tissue in order to obtain differentiated cells for various uses, and the potential for greater reproducibility and control of the process. With respect to pancreratic cells, successful achievement requires the differentiation and maturation of glucose-sensing, insulin-secreting beta cells from an expandable precursor population.